Elastic fibers are extracellular fibers responsible for the elasticity of highly flexible tissues such as the lungs, arteries, and skins. The major feature of human aging is a loss of tissue elasticity, which results in pulmonary edema, arterial sclerosis and snaking, skin loosening, and the like. These are increasingly important challenges in the aging society, many of which are caused by deterioration or rupture of elastic fibers. Despite the importance of elastic fibers, details of the molecular mechanisms of elastic fiber formation and deterioration remain unclear.
During the formation of elastic fibers, it is important that elastin deposits along fibers called microfibril and is crosslinked by enzymes of the lysyl oxidase family [lysyl oxidase (LOX), lysyl oxidase-like (LOXL) 1-4] [Molnar, J. et al., Biochim Biophys Acta 1647: 220-4 (2003); Rosenbloom, J. et al., Faseb J. 7: 1208-18. (1993)]. However, only a little is known about the molecular mechanisms based on which this process occurs in a living organism. Although microfibril is reported to essentially comprise long high-molecular proteins such as fibrillin 1, fibrillin 2, and LTBP2 (latent TGFb-binding protein 2), fibrillin 1 or fibrillin 2 gene knockout mice are free from elastic fiber abnormalities; therefore, contribution of these proteins to the formation of elastic fibers is unlikely [Pereira, L. et al., Nat. Genet. 17: 218-22 (1997), Putnam, E. A. et al., Nat. Genet. 11: 456-8 (1995), Chaudhry, S. S. et al., Mol. Genet. 10: 835-43 (2001)], and it remains unknown whether or not LTBP2 contributes to the formation of elastic fibers because LTBP2 gene knockout mice are fatal in early fetal period [Shipley, J. M. et al., Mol. Cell Biol. 20: 4879-87 (2000)].
The present inventors cloned a secretory protein known as DANCE (developmental arteries and neural crest epidermal growth factor (EGF)-like; also referred to as fibulin-5) using the signal sequence trap method [Nakamura, T. et al., J. Biol. Chem. 274: 22476-83 (1999)], prepared knockout mice lacking the expression of the protein, and found that elastic fibers in the whole body have been disjoined [Nakamura, T. et al., Nature 415: 171-5 (2002)]. For this reason, the phenotype of DANCE gene-deficient mice is highly similar to human aging, showing a lack of elasticity and loosening of the skin, severe pulmonary edema, and arterial tortuosity and sclerosis. Hence, DANCE is an essential protein for the formation of elastic fibers. Also, the present inventors have shown that the binding of DANCE to integrin can play an important role in living organisms [Nakamura, T. et al., J. Biol. Chem. 274: 22476-83 (1999)].
Recently, it was reported that knockout mice lacking the expression of LOXL1, one of the elastin-crosslinking enzymes, like DANCE knockout mice, exhibited abnormalities of the formation of elastic fibers [Liu, X. et al., Nat. Genet. 36: 178-82 (2004)]. Because LOXL1 binds to DANCE, and also because LOXL1 is no longer localized on elastic fibers in DANCE knockout mice, it is postulated that DANCE serves as an adapter to anchor the LOXL1 enzyme at a due position. Because the phenotype of LOXL1 knockout mice is weaker and emerges slightly later than the phenotype of DANCE knockout mice, the role of DANCE is considered to be more than anchoring LOXL1; the finding that DANCE defines the localization of the elastin-crosslinking enzyme is important in understanding the molecular mechanism by which DANCE contributes to the formation of elastic fibers.
However, in view of the fact that elastic fibers are formed along microfibril, it is considered that the binding of DANCE to the elastin-crosslinking enzyme is insufficient, and that DANCE needs to bind to a microfibril constituent protein. However, it remains unknown to which one of the microfibril proteins DANCE binds. Elucidating the detailed functions of DANCE is strongly demanded since it would enable the development of a pharmaceutical having a new mechanism of action enabling the regulation of the formation of elastic fibers.